HPTLC Analysis and Chemical Composition of Selected Melaleuca Essential Oils

Tea tree oil (TTO) is a volatile essential oil obtained by distillation, mainly from the Australian native plant Melaleuca alternifolia (Maiden & Betche) Cheel (Myrtaceae). In this study, a comparative analysis of the chemical constituents of seven tea tree oils (M. alternifolia) and four other Melaleuca spp. oils (M. cajuputi, (MCa), two chemotypes of M. quinquenervia, (MNe and MNi), and M. ericifolia (MRo)) was carried out using gas chromatography–mass spectrometry (GC-MS) and high-performance thin-layer chromatography (HPTLC). Among the seven TTOs, terpinen-4-ol (37.66–44.28%), γ-terpinene (16.42–20.75%), α-terpinene (3.47–12.62%), α-terpineol (3.11–4.66%), and terpinolene (2.75–4.19%) were the most abundant compounds. On the other hand, the most abundant compounds of the other Melaleuca oils varied, such as 1,8-cineole (64.63%) in MCa oil, (E)-nerolidol (48.40%) and linalool (33.30%) in MNe oil, 1,8-cineole (52.20%) in MNi oil, and linalool (38.19%) and 1,8-cineole (27.57%) in MRo oil. HPTLC fingerprinting of Melaleuca oils enabled the discrimination of TTO oils from other Melaleuca spp. oils. Variation was observed in the profile of the Rf values among EOs. The present study shows that HPTLC is one of the best ways to identify and evaluate the quality control in authenticating TTOs, other Melaleuca EOs, or EOs from other species within the Myrtaceae.

TTO and other Melaleuca EOs were subjected to PCA and HCA in order to identify which constituents (detected at ≥0.5%) are different among the TTOs and the four other Melaleuca species. Principle components are reflected by eigenvalues. Table 3 shows that the seven components with eigenvalues greater than one account for 96.35% of the total variance. According to the rules of PCA, the highest eigenvalues, F1 (14.92) and F2 (8.60), were selected and then subjected to the PCA analysis. The Bartlett s sphericity test carried out on the correlation matrix shows a calculated x 2 = 1519.98, greater than the critical value x 2 = 52.19 with 37 degrees of freedom (p < 0.0001), thus proving that PCA can achieve a significant reduction in the dimensionality of the original data set. The PCA plot established according to the first two PCA axes is shown in Figure 1 Table 1. The nomenclature of volatile compounds is listed in Table 2. Same color highlights similarity in chemical composition based on statistical analysis (See Section 3.5).
PCA provides a simple way to visualize similarities among different samples; a short range between the samples means a small or very little difference, and a long distance means a strong difference. Factor loadings and squared cosine (cos 2 ) indicate the importance of components representing the individual components for a given principal component ( Table 4) Table 1. The nomenclature of volatile compounds is listed in Table 2. Same color highlights similarity in chemical composition based on statistical analysis (See Section 3.5).
HCA classified Melaleuca EOs in two main groups ( Figure 2): group I clustered samples with high contents of terpinene-4-ol, γ-terpinene, α-terpinene, and terpinolene. Although M Ro did not belong to M. alternifolia (TTOs), it was grouped in cluster I due to its high levels of interfering compounds such as limonene, aromadendrene, and alloaromadendrene. The Euclidean distance between TTAA and TTPT was 1.74, and between TTAA and M Ro it was 7.51. Group II clustered samples M Ca , M Ni , and M Ne , which presented high contents of 1,8-cineole, linalool, and (E)-nerolidol, with intermediate values of α-pinene, αterpineol, and β-caryophyllene, and lower contents of terpinene-4-ol, γ-terpinene, terpineol, and α-terpinene, indicating that the TTOs contained significantly higher concentrations of terpinen-4-ol when compared to the other Melaleuca EOs.  Table 1. The color box indicated the abundance of each compound. Red represents high density and blue represents low density.

HPTLC Analysis of Melaleuca EOs
The less-polar components of the UHM, labeled (e) through (h) according to Do et al. [38], separated well under the selected HPTLC development conditions, as shown in Figure 3A. This indicated that the initial method involving Hex/EtOAc 90:10 (v/v) as the solvent system was appropriate for the separation of non-polar components of interest in our samples. However, the Rf of some Melaleuca oil components appear to exceed that of I II Figure 2. Two-way dendrogram of the hierarchical cluster analysis (HCA) performed on the chemical composition of the seven tea tree (M. alternifolia) EOs and four other Melaleuca EOs. Sample codes refer to Table 1. The color box indicated the abundance of each compound. Red represents high density and blue represents low density.

HPTLC Analysis of Melaleuca EOs
The less-polar components of the UHM, labeled (e) through (h) according to Do et al. [38], separated well under the selected HPTLC development conditions, as shown in Figure 3A. This indicated that the initial method involving Hex/EtOAc 90:10 (v/v) as the solvent system was appropriate for the separation of non-polar components of interest in our samples. However, the R f of some Melaleuca oil components appear to exceed that of the highest UHM component. For future purposes, an additional component with higher R f may need to be added to the UHM to better encompass the range of our samples.
In general, the HPTLC results complemented the findings obtained from the GC-MS analysis. The oils of M. alternifolia (TTOs) (Figure 3B), despite their wide variety of sources, exhibited a very similar separation pattern dominated by terpinen-4-ol as the primary component. When developed with Hex/EtOAc 90:10 (v/v), this main constituent of TTO appeared at an R f value of 0.278 ± 0.067. A group of mono-and sesquiterpenes, including α-and β-pinene (R f = 0.767 ± 0.031) and (+)-aromadendrene (R f = 0.738 ± 0.062), was showcased as the second most prominent band, with a collective R f of 0.747 ± 0.065. Included in this group were ledene and δ-cadinene, identified by GC-MS. This band was followed in intensity by α-terpineol (R f = 0.147 ± 0.052) and 1,8-cineole (R f = 0.504 ± 0.081), respectively.
In cases where oil components were merged, as observed by the overlapping or blending of colors, such components exhibited a shift in R f values compared to those of the corresponding reference standards. This may be caused by interactions due to large amounts of components competing for the limited silica surface area. The R f of the merged oil constituents, as well as the presence of additional constituents, was established by GC-MS.
Small, yet still characteristic of M Ne , was geraniol at R f = 0.124 ± 0.067. A bright-pink band at R f = 0.432 ± 0.045 was recognized as caryophyllene oxide by isolating the component on a TLC preparation plate (Section 3.4.2) and confirmed by GC-MS analysis against its reference standard and available GC-MS libraries. Caryophyllene oxide appeared to be a byproduct of β-caryophyllene since it was present in both TLC and TIC reference standard chromatograms.
The M Ni oil was characterized by a large amount of 1,8-cineole. As with M Ca , this large band was mixed with a small amount of terpinyl acetate and limonene. The second-largest band was composed of mono-and sesquiterpenes, confirmed by GC-MS as αand β-pinene, β-caryophyllene, and ledene. Other main components were nerolidol, α-terpineol, and viridiflorol (R f = 0.213 ± 0.021).
In M Ro , the signature component was linalool, with a small amount of terpinen-4-ol merging into it. Mono-and sesquiterpenes composed the second-strongest band near 0.76 R f , followed by cineole as the third characteristic band. Constituents of the second band were recognized as α-pinene, aromadendrene, alloaromadendrene, and ledene by GC-MS. Merged into a fourth band was α-terpineol, followed by globulol a bit below, at R f = 0.137 ± 0.022. System suitability as well as R f values for the major components were established by SSTs ( Figure 4A) and reference standards ( Figure 4B), analyzed under the same HPTLC conditions. and the individual reference standards ( Figure 6B) analyzed under exact HPTLC developing conditions.  Table 5), tea tree oils (B), and other Melaleuca oils (C) (see Table 1) under visible light, developed with Hex/EtOAc 90:10 (v/v) on Silica gel 60 F 254.      Table 5), tea tree oils (B), and other Melaleuca oils (C) (see Table 1) under visible light, developed with Hex/EtOAc 90:10 (v/v) on Silica gel 60 F 254 .    Table 5), tea tree oils (B), and other Melaleuca oils (C) (see Table 1) under visible light, developed with Hex/EtOAc 90:10 (v/v) on Silica gel 60 F 254.
An enhanced separation of more polar constituents in the oils (lower R f range) was obtained when developed with Hex/EtOAc at a ratio of 80:20 (v/v). Figure 5A emphasizes an increased distance among UHM constituents. Additionally, target oil components that merged at low R f values under the previous solvent system were now appearing mid-range and better separated.

Sample Selection and Preparation
TTOs from M. alternifolia were selected based on their previously established biological activity as a potential attractant for the male Mediterranean fruit fly [23,24,26,27]. Essential oils from other Melaleuca species were also included for the purpose of comparison (Table 1). Each sample was diluted to 20% of its original purity using methylene chloride, ACS Reagent, CAS# 75-09-2 (J.T. Baker-Avantor, Center Valley, PA, USA). If necessary, concentration and application volume were adjusted for optimum HPTLC separation.

Standard Selection and Preparation
A universal HPTLC calibration mix (UHM) (Sigma-Aldrich, St. Louis, MO, USA) was used as a reference standard for HPTLC separations. It contains eight different compounds diluted in methanol at ready-to-use concentrations [38], four of which were observed under our HPTLC conditions using a UV 254 light source (Table 5).
A series of reference standards (Table 6) were obtained from Sigma-Aldrich (St. Louis, MO, USA), and were prepared and analyzed under the same conditions as the EOs to confirm the R f values of the oil components on HPTLC.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
Melaleuca EO samples were analyzed on an Agilent 7890B GC coupled with a 5977B mass selective detector (GC-MS) (Agilent Technologies, Santa Clara, CA, USA). A DB-5 column (30 m × 0.25 mm inner diameter with 0.25 µm film thickness) was used with an electron ionization source set at 70 eV. The temperatures of the ion source and quadrupole were 230 • C and 150 • C, respectively. The mass spectrometry transmission line was 250 • C. Injector and detector temperatures were kept at 220 • C and 230 • C, respectively. The oven temperature program was set at 60 • C for 1.3 min and increased to 246 • C at 3 • C/min. A constant helium flow of 1.3 mL/min was applied [41]. The selected mass range was m/z 35 to 450 Da and scan rate was 2.8 scans/s. Mass Hunter B.07.06 software (Agilent Technologies) was used for data acquisition and processing. One µL of diluted samples was injected into the GC-MS on splitless mode.
Linear retention indices (RIs) were calculated using the van Den Dool and Kratz [42] equation in relation to a homologous series of n-alkanes (C 9 -C 21 ). Compound identification was achieved by comparison of their corresponding mass spectra and RIs to those reported in a mass spectral library developed at the USDA-ARS-SHRS laboratory with authentic compounds and with the commercial libraries MassFinder [43], Adams Library [41], Flavours and Fragrances of Natural and Synthetic Compounds 3 (FFNSC-3) [44], Wiley 12/NIST 2020 [45], and an in-house library "SHRS Essential Oil Constituents-DB-5 Column". Retention indices were also verified with data reported in the specific literature [46][47][48][49][50][51][52][53][54] and internet sources [55][56][57]. Each oil was analyzed in triplicate. Relative percentages were directly obtained from peak total ion current (TIC) areas. All these standards were purchased from the following sources Chromatography was performed using a CAMAG HPTLC system equipped with VisionCATS 3.1 software (CAMAG, Muttenz, Switzerland). Initial conditions were set following the established HPTLC/TLC protocol for essential oils [37,63]. An HPTLC Silica gel 60 F 254 glass-backed plate, 20 × 10 cm (Supelco Merck KGaA, Darmstadt, Germany, operating as Millipore-Sigma in St. Louis, MO, USA), was activated by heat using a TLC Plate Heater III (CAMAG, Muttenz, Switzerland) for 10 min. at 65 • C prior to analysis. Toluene/ethyl acetate 93:7 (v/v) was used as the mobile phase. However, previous bioassays (P.E.K. unpublished data) had indicated that sterile male medflies were repelled by toluene residue, prompting the search for an alternative mobile phase. Hexane was selected due to its similar polarity to toluene.
Melaleuca EO constituents appear in different amounts and cover a relatively wide polarity range when separated by TLC. Tabanca et al. [27] used hexane/acetone 90:10 (v/v) and obtained a good separation that produced two TTO fractions attractive to sterile male medflies, yet these fractions still contained a mixture of chemicals. Further separation was necessary to identify possible individual attractants. Various ratios of hexane/ethyl acetate were then attempted in preliminary experiments and it was decided that two separate solvent combinations provided improved resolution of the fractions of interest.
An aliquot of each oil sample was dispensed into a 1.5 mL screw-cap vial, covered with TFE/SIL septum cap (J.G. Finneran Associates, Inc., Vineland, NJ, USA), and placed into an Automatic TLC Sampler (ATS4) (CAMAG, Muttenz, Switzerland). An activated silica gel plate was placed in its corresponding holder. Samples were applied as thin bands (8 mm long, 8 mm from the bottom edge of the plate) using a 25 µL Hamilton syringe with spray application needle and nozzle. Syringe and needle were automatically rinsed 5 times with methanol, ACS grade, CAS# 67-56-1 (Supelco Merck KGaA, Darmstadt, Germany, as EMD Millipore Corporation, Burlington, MA, USA), between samples.
The HPTLC plate was developed in an Automatic Developing Chamber (ADC2) (CAMAG, Muttenz, Switzerland). The chamber containing a saturation pad was saturated for 20 min with 25 mL of the selected mobile phase. To remove as much moisture as possible, the system was also activated for 10 min with a saturated magnesium chloride aqueous solution prepared from magnesium chloride hexahydrate, (MgCl 2 .6H 2 O), CAS# 7791-18-6 (Sigma-Aldrich, St. Louis, MO, USA). Development was automatically started and stopped once the solvent front reached a preset height of 85 mm. After development, the plate was allowed to dry for 1 min at room temperature in the fume hood.
Automatic derivatization of the plate to generate color occurred inside a Derivatizer chamber (CAMAG, Muttenz, Switzerland) with 2 mL of vanillin/H 2 SO 4 reagent. The derivatizing reagent was applied by spraying through a yellow nozzle at spray level 3. Colors were observed after heating the plate for 1.5 to 3.0 min at 100 • C on a CAMAG Plate Heater III, depending on color intensity. Images of the plate were taken at various stages of the process using a Visualizer 2 (CAMAG, Muttenz, Switzerland) with a 16 mm lens under RT White, UV 254 , and UV 366 light. The retention factors (R f ) values were calculated by VisionCATS software version 3.1. Profiles and comparisons were also generated using VisionCATS.

Automated Preparative Thin-Layer Chromatography Analysis
Preparative TLC was used to isolate unknown bands for identification in cases where a component was not readily identified, and a standard could not be easily referenced for confirmation. An HPTLC Silica gel 60 F 254 glass-backed plate, 20 × 10 cm (Supelco Merck KGaA, Darmstadt, Germany, operating as Millipore-Sigma in St. Louis, MO, US), was used to separate and collect a reasonable amount of the unknown chemical for further identification. The plate was initially activated by heating at 100 • C for 15 min on a CAMAG TLC Plate Heater III.
Sample application was conducted in a CAMAG ATS4 autosampler, where 2 µL oil was applied in 20 consecutive bands, 8 mm long each, making a solid horizontal line 8 mm from the bottom edge of the plate. Once the sample was applied, the plate was developed in a previously saturated CAMAG ADC2 chamber. A total of 35 mL mobile phase was used, 25 mL for saturation and 10 for development. Development stopped when solvent front reached 85 mm.
In the case of nerolina oil, a bright pink band at R f = 0.451 ± 0.022 (40-45 mm from the bottom) was our target chemical. A 5 × 10 cm strip was cut out of the developed plate and was sprayed with vanillin reagent in a CAMAG Derivatizer. The bright pink band on the derivatized strip provided the measurements of the area to scrape to obtain our unknown from the remaining (non-derivatized) portion of the plate. Scraped silica containing our compound of interest was extracted with 1 mL methylene chloride and filtered through a 0.2 µm Whatman AUTOVIAL™ 5 syringeless filter (Global Life Sciences Solutions USA LLC-Cytiva, Marlborough, MA, USA) for GC-MS analysis.

Statistical Analysis
Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to TTOs and other Melaleuca EOs and their chemical constituents, using the XLSTAT 2021 (Addinsoft, New York, NY, USA) for PCA and JMP (JMP ® Pro 17.0.0, SAS Institute Inc. Cary, NC, USA) for HCA. Both PCA and HCA were performed on the means of those volatile constituents higher than 0.5%; the covariance data matrix was 38 × 11 (418 data). Pearson's correlation model was used for PCA, Euclidean distance for measure, and Ward's method for HCA analysis.

Conclusions
The results of this study demonstrate that HPTLC serves as a quick and effective analytical technique for the screening of selected Melaleuca oils. Its automated steps eliminate most human error and provide better reproducibility. A wide variety of samples may be analyzed by combining the most suitable mobile and stationary phases for the target analytes. This allows for the selection of less toxic solvents, such as hexane instead of toluene, while maintaining comparable retention factors to those in the Pharmacopeia. It also provides a fast detection tool for more polar additives or contaminants that may not be detected under GC-MS conditions. Samples can be applied as a long, narrow band, allowing multiple samples to be simultaneously analyzed and a cleaner separation of individual components.
An advantage over other analytical techniques is that multiple samples and standards may be analyzed at the same time and under true identical conditions using HPTLC. Moreover, the development process is nondestructive, which allows samples to be scraped and extracted from the plate for further studies. For this purpose, a template may be created from a prior plate derivatized with color reagent.
On the other hand, there are some disadvantages to this procedure. In the case of highly volatile constituents, there is a high probability of evaporation during the process. In addition, some oil components do not react with the derivatizing reagent and therefore do not emit a visible color. While some may be seen under UV light, others may not be visible at all. Another complicating factor is that compounds found in trace amounts may fall under the detection limit of the HPTLC instrument. A more complex mixture of coeluting chemicals also represents a challenge. Two or more developments may be required for better separation of these target constituents.
New studies are currently in process, involving two-dimensional and multigradient developments to address the above-mentioned challenges. Future work prospects include the addition of a densitometry module and a TLC-MS interface to achieve a more accurate quantification and precise recovery of individual oil components for further analysis. With so many favorable features and few obstacles, this technique proved to be an efficient and reliable screening tool for the selected Melaleuca oils.